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MACS separation columns, LS

MS色谱柱

Company: Miltenyi Biotec
Catalog#: 130-042-401
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Generation of T cells from Human and Nonhuman Primate Pluripotent Stem Cells
Author:
Date:
2020-07-05
[Abstract]  Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally ... [摘要]  [摘要] 多能干细胞(PSCs)有潜力提供同质的T细胞群体,这些细胞可以在临床规模上生长,并通过基因工程来满足特定的临床需求。OP9-DLL4是一种异位表达Notch配体Delta-like 4(DLL4)的基质细胞系,用于支持psc向T淋巴细胞的分化。本文概述了从人类和非人类灵长类(NHP)PSC中产生T细胞的几种方法,包括在OP9喂食者或特定条件下对PSC进行初始造血分化,然后将OP9-DLL4细胞与PSC衍生的造血祖细胞(HPs)共培养,从而有效地分化成T淋巴细胞。此外,我们描述了一个从有条件表达ETS1的hPSCs中产生健壮T细胞的方案。所提出的协议提供了一个平台,用于疾病建模和评估其在大型动物模型免疫治疗中的应用。

[背景] T淋巴细胞(T细胞)在细胞介导的免疫反应中起着关键作用,参与肿瘤细胞的监测和杀伤。在过去的几十年里,已经开发了几种策略来重定向、培养和/或增强抗肿瘤的T淋巴细胞(Houot等人,2015年;June等人,2018年),并将其用于基于T细胞的过继免疫治疗。最近的临床试验表明,用嵌合抗原受体(CAR)-T细胞治疗复发性和难治性淋巴瘤患者的疗效显著(Riviere和Sadelain,2017)。

人类多能干细胞(hPSCs),包括胚胎(hESCs)和诱导(hiPSCs),为生产用于过继性细胞免疫疗法的T细胞提供了一种很有前景的资源,可与基因工程技术相结合,产生现成的CAR ...

Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice
Author:
Date:
2020-05-20
[Abstract]   T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in ... [摘要]  [摘要 ] T细胞急性淋巴细胞白血病(T-ALL)是一种侵袭性血液恶性肿瘤,其起源于T细胞引发的造血祖细胞的转化。尽管T-ALL是一种异质且分子复杂的疾病,但超过65%的T-ALL患者在NOTCH1 基因中带有激活突变。大多数与T-ALL相关的NOTCH1 突变要么破坏负调控区,允许在没有配体结合的情况下激活信号,要么导致蛋白酶体降解终止NOTCH1信号终止所涉及的C末端PEST域被截短。迄今为止,逆转录病毒转导模型在很大程度上依赖于侵袭性截短的变种的过度表达。 NOTCH1 (例如ICN1或ΔE-NOTCH1)可导致信号传导的超生理水平,并且在人类T-ALL中很少见。当前方案描述了小鼠骨髓分离,造血干细胞和祖细胞(HSC)富集,然后逆转录病毒转导的致癌突变体形式的NOTCH1受体(NOTCH1-L1601P-ΔP)的方法,该方法与获功能突变最常见于患者样品中。组成型活性NOTCH1的这种强制表达的标志是胸腺外未成熟T细胞发育的瞬时波,此波在致癌性转化为T-ALL之前。此外,该方法通过允许白血病细胞与微环境之间的串扰来模拟体内白血病的转化和进展,这是基于细胞系的体外研究无法解释的一个方面。因此,HSC转导和移植模型更真实地概括了人类疾病的发展,为进一步的体内和离体功能研究提供了高度全面和通用的工具。

[背景 ] ...

Primary Cultures from Human GH-secreting or Clinically Non-functioning Pituitary Adenomas
Author:
Date:
2018-04-05
[Abstract]  Pituitary adenomas are among the more frequent intracranial tumors usually treated with both surgical and pharmacological–based on somatostatin and dopamine agonists–approaches. Although mostly benign tumors, the occurrence of invasive behaviors is often detected resulting in poorer prognosis. The use of primary cultures from human pituitary adenomas represented a significant advancement in the knowledge of the mechanisms of their development and in the definition of the determinants of their pharmacological sensitivity. Moreover, recent studies identified also in pituitary adenomas putative tumor stem cells representing, according to the current hypothesis, the real cellular targets to eradicate most malignancies. In this protocol, we describe the procedure to establish primary cultures ... [摘要]  垂体腺瘤是更常见的颅内肿瘤之一,通常用基于生长抑素和多巴胺激动剂手术的手术和药物治疗。 虽然多为良性肿瘤,但侵入性行为的发生常常被检测到,导致预后较差。 来自人类垂体腺瘤的原代培养物的使用代表了对其发育机制的知识以及其药理敏感性决定因素的定义方面的显着进步。 此外,最近的研究也在垂体腺瘤中发现了假定的肿瘤干细胞,根据目前的假设,它代表了根除大多数恶性肿瘤的真实细胞靶标。 在这个协议中,我们描述了从人垂体腺瘤建立原代培养的程序,以及如何选择,体外扩增和表型鉴定推定的垂体腺瘤干细胞。

【背景】垂体腺瘤是最常见的颅内肿瘤之一(高达15%),横断面研究发现每100,000名居民中约有90例发病,其中绝大多数为30岁以上的成年人。大约10%的未经选择的垂体在尸检时进行了检查(即考虑到之前未诊断为垂体疾病的受试者的垂体)( ,Molitch,2017)。尽管通常为良性肿瘤,但垂体腺瘤的处理可因与激素分泌过多相关的临床综合征或发展以治疗抗性,高增殖率,快速复发和绒毛外侵袭为特征的侵袭行为而复杂化(Carreno等人,2017)。成年垂体干细胞的持续存在(Florio,2011)导致垂体腺瘤(以及可能的其他良性瘤形成)的发展可以源自具有干细胞特性(主要是自我更新和分化)的肿瘤细胞的亚群能力),正如已经建立的恶性固体和血液肿瘤一样。

最近的实验证据表明,癌症干细胞(CSC)范例也适用于人和小鼠垂体腺瘤(Donangelo等人,2014; ...

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